anandamide and Breast-Neoplasms

Evidence for the role of the cannabimimetic fatty acid derivatives (CFADs), i.e. anandamide (arachidonoylethanolamide, AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA), in the control of inflammation and of the proliferation of tumor cells is reviewed here. The biosynthesis of AEA, PEA, or 2-AG can be induced by stimulation with either Ca(2+) ionophores, lipopolysaccharide, or platelet activating factor in macrophages, and by ionomycin or antigen challenge in rat basophilic leukemia (RBL-2H3) cells (a widely used model for mast cells). These cells also inactivate CFADs through re-uptake and/or hydrolysis and/or esterification processes. AEA and PEA modulate cytokine and/or arachidonate release from macrophages in vitro, regulate serotonin secretion from RBL-2H3 cells, and are analgesic in some animal models of inflammatory pain. However, the involvement of endogenous CFADs and cannabinoid CB(1) and CB(2) receptors in these effects is still controversial. In human breast and prostate cancer cells, AEA and 2-AG, but not PEA, potently inhibit prolactin and/or nerve growth factor (NGF)-induced cell proliferation. Vanillyl-derivatives of anandamide, such as olvanil and arvanil, exhibit even higher anti-proliferative activity. These effects are due to suppression of the levels of the 100 kDa prolactin receptor or of the high affinity NGF receptors (trk), are mediated by CB(1)-like cannabinoid receptors, and are enhanced by other CFADs. Inhibition of adenylyl cyclase and activation of mitogen-activated protein kinase underlie the anti-mitogenic actions of AEA. The possibility that CFADs act as local inhibitors of the proliferation of human breast cancer is discussed here.

Topics: Adjuvants, Immunologic; Amides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Arachidonic Acids; Breast Neoplasms; Cannabinoids; Cell Division; Endocannabinoids; Ethanolamines; Glycerides; Humans; Inflammation; Male; Neoplasms; Palmitic Acids; Polyunsaturated Alkamides; Prostatic Neoplasms; Rats; Receptors, Growth Factor

The present study was carried out to investigate anti-tumoral effects of Anandamide (AEA) in luminal A breast cancer cell line MCF-7. Cell viability was measured by MTT assay and cell index was measured by xCelligence DP analyzer system. The Feulgen method was used to determine the mitotic index parameter, and the 3H-Thymidine method was used to determine the labeling index parameter. The apoptotic index parameter was determined using a fluorescent dye DAPI. The results of this study showed that 25 µM Anandamide concentration was the optimum concentration for MCF-7 cells. While this concentration decreased the proportion of cells in the mitotic phase and synthesis phase, it increased the proportion of apoptotic cells.

Topics: Arachidonic Acids; Breast Neoplasms; Cannabinoids; Endocannabinoids; Female; Humans; MCF-7 Cells; Polyunsaturated Alkamides

Topics: Arachidonic Acids; beta Catenin; Breast Neoplasms; Cannabinoid Receptor Agonists; Cell Line, Tumor; Cell Proliferation; Endocannabinoids; Epithelial-Mesenchymal Transition; Female; Humans; Polyunsaturated Alkamides; Wnt Signaling Pathway

Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in NF (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway.. The aim of this study was to analyse the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signalling pathway and heme oxygenase-1 (HO-1) induction and transcription.. Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034 ng per 10(6) cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Because breast tumour cells express FAAH abundantly, we examined the effects of FAAH on Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent of the cannabinoid receptors, CB1, CB2 or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells.. These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signalling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors.

Topics: Active Transport, Cell Nucleus; Amidohydrolases; Antineoplastic Agents; Arachidonic Acids; Benzamides; Breast Neoplasms; Carbamates; Cell Survival; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Induction; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Heme Oxygenase-1; Humans; MCF-7 Cells; NF-E2-Related Factor 2; Polyunsaturated Alkamides; RNA Interference; Signal Transduction; Transcription, Genetic; Transfection

The endocannabinoid system regulates cell proliferation in human breast cancer cells. Recently, we described that a metabolically stable anandamide analog, 2-methyl-2'-F-anandamide, by activation of CB1 receptors significantly inhibited cell proliferation of human breast cancer cell lines. In this study, we observed that the activation of the CB1 receptor, in two human mammary carcinoma cell lines, MDA-MB-231 and MCF7, caused the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity due to a reduction of HMG-CoA reductase transcript levels. The decrease of HMG-CoA reductase activity induced the inhibition of the prenylation of proteins, in particular of the farnesylation of Ras oncogenic protein involved in cell proliferation of these cell lines. We suggest that the inhibitory effect of anandamide analog on tumor cell proliferation could be related to the inhibition of Ras farnesylation.

Topics: Alkyl and Aryl Transferases; Antineoplastic Agents; Arachidonic Acids; Breast Neoplasms; Cannabinoid Receptor Modulators; Carcinoma; Cell Line, Tumor; Down-Regulation; Drug Evaluation, Preclinical; Endocannabinoids; Enzyme Inhibitors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Polyunsaturated Alkamides; ras Proteins

The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB1 receptors could induce a non-invasive phenotype in breast metastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.

Topics: Animals; Apoptosis; Arachidonic Acids; Breast Neoplasms; Cannabinoid Receptor Modulators; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Endocannabinoids; Female; Focal Adhesion Kinase 1; Humans; Integrins; Lung Neoplasms; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; src-Family Kinases; Time Factors

This study was designed to determine the molecular mechanisms underlying the anti-proliferative effect of the endocannabinoid anandamide on highly invasive human breast cancer cells, MDA-MB-231. We show that a metabolically stable analogue of anandamide, Met-F-AEA, induces an S phase growth arrest correlated with Chk1 activation, Cdc25A degradation and suppression of Cdk2 activity. These findings demonstrate that Met-F-AEA induced cell cycle blockade relies on modulated expression and activity of key S phase regulatory proteins. The observed mechanism of action, already reported for well-known chemotherapeutic drugs, provides strong evidence for a direct role of anandamide related compounds in the activation of cell cycle checkpoints.

Topics: Arachidonic Acids; Breast Neoplasms; cdc25 Phosphatases; Cell Cycle; Cell Line, Tumor; Checkpoint Kinase 1; Cyclin-Dependent Kinase 2; Endocannabinoids; Female; Humans; Polyunsaturated Alkamides; Protein Kinases; S Phase; Tumor Cells, Cultured

In this report we show, by confocal analysis of indirect immunofluorescence, that the type-1 cannabinoid receptor (CB1R), which belongs to the family of G-protein-coupled receptors, is expressed on the plasma membrane in human breast cancer MDA-MB-231 cells. However, a substantial proportion of the receptor is present in lysosomes. We found that CB1R is associated with cholesterol- and sphyngolipid-enriched membrane domains (rafts). Cholesterol depletion by methyl-beta-cyclodextrin (MCD) treatment strongly reduces the flotation of the protein on the raft-fractions (DRM) of sucrose density gradients suggesting that CB1 raft-association is cholesterol dependent. Interestingly binding of the agonist, anandamide (AEA) also impairs DRM-association of the receptor suggesting that the membrane distribution of the receptor is dependent on rafts and is possibly regulated by the agonist binding. Indeed MCD completely blocked the clustering of CB1R at the plasma membrane. On the contrary the lysosomal localization of CB1R was impaired by this treatment only after AEA binding.

Topics: Arachidonic Acids; beta-Cyclodextrins; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cholesterol; Endocannabinoids; Humans; Lysosomes; Membrane Microdomains; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

We have earlier reported overexpression of the central and peripheral cannabinoid receptors CB1 and CB2 in mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma. In this study, treatment with cannabinoid receptor ligands caused a decrease in viability of MCL cells, while control cells lacking CB1 were not affected. Interestingly, equipotent doses of the CB1 antagonist SR141716A and the CB1/CB2 agonist anandamide inflicted additive negative effects on viability. Moreover, treatment with the CB1/CB2 agonist Win-55,212-2 caused a decrease in long-term growth of MCL cells in culture. Induction of apoptosis, as measured by FACS/Annexin V-FITC, contributed to the growth suppressive effect of Win-55,212-2. Our data suggest that cannabinoid receptors may be considered as potential therapeutic targets in MCL.

Topics: Animals; Arachidonic Acids; Benzoxazines; Biopsy; Breast Neoplasms; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cell Death; Cell Division; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cells, Cultured; Dose-Response Relationship, Drug; Endocannabinoids; Female; Flow Cytometry; Humans; Leukemia, Plasma Cell; Ligands; Lymphoma, Mantle-Cell; Mice; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Rimonabant

Topics: Animals; Arachidonic Acids; Breast Neoplasms; Cannabinoids; Cell Division; Eicosanoids; Endocannabinoids; Female; Glycerides; Humans; Inflammation; Polyunsaturated Alkamides; Rats; Rats, Wistar; Tumor Cells, Cultured; Urinary Bladder

Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.

Topics: Amides; Amidohydrolases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Arachidonic Acids; Blotting, Western; Breast Neoplasms; Camphanes; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Cell Division; Colforsin; COS Cells; Cyclic AMP; Dose-Response Relationship, Drug; Endocannabinoids; Ethanolamines; Glycerides; Humans; Hydrolysis; Inhibitory Concentration 50; Palmitic Acids; Polyunsaturated Alkamides; Protein Binding; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured

Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. Beta-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50-600 nM) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB2 receptor antagonist, SR144528. Anandamide and HU-210 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC50 = 100-300 nM) by anandamide, 2-AG, and HU-210. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB2 cannabinoid receptors, 2) specific binding sites for [3H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein. These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.

Topics: Arachidonic Acids; Binding Sites; Blotting, Western; Breast Neoplasms; Cannabinoid Receptor Modulators; Cannabinoids; Cell Division; Endocannabinoids; Female; Glycerides; Humans; Male; Neoplasms, Hormone-Dependent; Nerve Growth Factors; Piperidines; Polyunsaturated Alkamides; Prostatic Neoplasms; Pyrazoles; Receptor Protein-Tyrosine Kinases; Receptors, Cannabinoid; Receptors, Drug; Receptors, Nerve Growth Factor; Receptors, Prolactin; Rimonabant; Tumor Cells, Cultured

Anandamide (ANA) inhibits prolactin- and nerve growth factor (NGF)-induced proliferation of human breast cancer cells by decreasing the levels of the 100 kDa prolactin receptor (PRLr) and the high affinity trk NGF receptor, respectively, and by acting via CB(1)-like cannabinoid receptors. However, the intracellular signals that mediate these effects are not known. Here, we show that, in MCF-7 cells: (i) forskolin and the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 prevent, and the protein kinase A inhibitor RpcAMPs mimics, the inhibitory effects of ANA on cell proliferation and PRLr/trk expression and (ii) ANA inhibits forskolin-induced cAMP formation and stimulates Raf-1 translocation and MAPK activity, in a fashion sensitive to the selective CB(1) antagonist SR141716A. ANA stimulation of MAPK was enhanced by inhibitors of ANA hydrolysis. Forskolin inhibited MAPK and ANA-induced Raf-1 translocation. These findings indicate that, in MCF-7 cells, ANA inhibits adenylyl cyclase and activates MAPK, thereby exerting a down-regulation on PRLr and trk levels and a suppression of cell proliferation.

Topics: Arachidonic Acids; Breast Neoplasms; Cell Division; Cell Line; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Endocannabinoids; Flavonoids; Humans; Mitogen-Activated Protein Kinases; Nerve Growth Factor; Piperidines; Polyunsaturated Alkamides; Prolactin; Proto-Oncogene Proteins c-raf; Pyrazoles; Receptors, Prolactin; Rimonabant; Tumor Cells, Cultured

Anandamide was the first brain metabolite shown to act as a ligand of "central" CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 microM and 83-92% maximal inhibition at 5-10 microM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 microM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55, 940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1-0.5 microM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.

Topics: Arachidonic Acids; Breast Neoplasms; Calcium Channel Blockers; Cannabinoids; Cell Division; Dose-Response Relationship, Drug; Endocannabinoids; Female; Humans; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Tumor Cells, Cultured

The endogenous cannabinoid, anandamide (arachidonoylethanolamide), and the sleep-inducing factor, oleamide (cis-9-octadecenoamide), represent two classes of long-chain fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human breast carcinoma EFM-19 cells and rat adrenal pheochromocytoma PC-12 cells, together with the enzyme responsible for their degradation, fatty acid amide hydrolase, and the proposed biosynthetic precursors for arachidonoylethanolamide and related acylethanolamides, the N-acyl-phosphatidylethanolamines. Lipids extracted from cells labelled with [14C]ethanolamine contained radioactive compounds with the same chromatographic behaviour as arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with ionomycin in EFM-19 cells or two-week treatment with the nerve growth factor in PC-12 cells. The chemical nature of arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of oleamide. The latter compound, which does not activate the central CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than arachidonoylethanolamide (IC50 = 11.3 microM for oleamide and 2.1 microM for arachidonoylethanolamide), while at a low, inactive dose it potentiated an arachidonoylethanolamide cytostatic effect. The CB1 receptor selective antagonist SR 141716A (0.5 microM) reversed the effect of both arachidonoylethanolamide and oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [14C]arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for fatty acid amide hydrolase. This enzyme was inhibited by oleamide in both intact cells and cell-free preparations. The presence of transcripts of fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total RNA. The rate of [14C]arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of arachidonoylethanolamide enzymatic hydrolysis and the amounts of the fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with nerve growth factor and subsequent transformation of PC-12 ce

Topics: Amidohydrolases; Animals; Arachidonic Acids; Breast Neoplasms; Cell Differentiation; Cell Division; Endocannabinoids; Humans; Oleic Acids; PC12 Cells; Pheochromocytoma; Phosphatidylethanolamines; Polyunsaturated Alkamides; Rats; RNA, Messenger; Tumor Cells, Cultured